TSA305 gene

ABSTRACT

The invention provides a pancreas-specific gene comprising a base sequence coding for the amino acid sequence shown under SEQ ID NO:1, which gene is effective particularly in the fields of study, diagnosis and treatment, among others, of pancreatic carcinoma.

TECHNICAL FIELD

The present invention relates to a gene, named TSA305, coding for aprotein specifically expressed in the pancreas and, more particularly,to the above pancreas-specific gene having a high level of homology withnematode sel-1 and expected to show an anticancer activity. Theinvention also relates to a novel protein encoded by such gene and to aspecific antibody thereto.

BACKGROUND ART

Pancreatic cancer holds the fourth and fifth place on the list ofcancer-related deaths in Japan and western countries, respectively andhas the worst prognosis among digestive system malignancies (Poston, J.G., et al., Gut, 32, 800-812 (1991)). The ultimate goal in cancerresearch is to discriminate early stage gene changes leading tomalignant transformation. If such changes can be differentiated, genetictools for early diagnosis may possibly be developed and noveltherapeutic approaches for more effective treatment of this lethaldisease will possibly be taken.

Meanwhile, the nematode sel-1 gene reportedly has an inhibitory actionon Notch/lin-12 which suppresses the differentiation of ectoderm intoneuroblast in neural development in nematodes (Genetics, 143 (1),237-247 (1996); Development, 124 (3), 637-644 (1997)). SaidNotch/lin-12, when forcedly expressed, causes breast cancer or leukemiaand therefore is considered to be a cancer-related gene. The above sel-1gene suppressively acting on said cancer-related gene is thereforeconsidered to suppressively act on cancer as well. At present, however,the roles of these genes have not been fully elucidated.

Elucidation of the physiological roles of such genes and the informationobtained therefrom are important in elucidating the mechanisms of onsetof diseases such as malignant transformation and inflammation and aredesired not only in the field of basic scientific studies but also inthe pharmaceutical field in determining the causes of such diseases ascancer and inflammation and developing treatment methods for suchdiseases.

DISCLOSURE OF INVENTION

The present invention has for its object to provide the aboveinformation desired in the relevant field of art, in particular a genecoding for a novel protein homolog and having homology with the sel-1gene.

With that object in view, the present inventors made an arduous searchamong genes derived from various human tissues and, as a result,succeeded in newly isolating and identifying a gene coding for a proteinspecifically expressed in the pancreas and found that the above objectcan be realized with said gene. As a result, the present invention hasnow been completed.

Thus, the present invention provides a pancreas-specific gene, TSA305,comprising a nucleotide sequence coding for a protein having the aminoacid sequence shown under SEQ ID NO:1, in particular the TSA305 genewhich is a human gene.

The invention also provides a pancreas-specific protein (TSA305 protein)comprising the amino acid sequence shown under SEQ ID NO:1 and anantibody capable of coupling therewith.

The invention further provides a pancreas-specific gene, TSA305, whichis a polynucleotide defined below under (a) or (b) in particular to theTSA305 gene which is a human gene:

(a) A polynucleotide comprising the whole or part of the nucleotidesequence shown under SEQ ID NO:2.

(b) A polynucleotide capable of hybridizing with a DNA having thenucleotide sequence shown under SEQ ID NO:2 under stringent conditions.

In addition, the present invention provides the above gene in DNAfragment form which is useful as a specific probe or specific primer forgene detection.

In expressing amino acids, peptides, nucleotide sequences, nucleic acidsand the like by abbreviations or symbols in the following, thenomenclature of the IUPAC-IUB [IUPAC-IUB Communication on BiologicalNomenclature, Eur. J. Biochem., 138: 9 (1984)], the “Guideline forpreparing specifications etc. containing nucleotide sequences or aminoacid sequences” (edited by the Patent Office of Japan) and theconventional symbols in the relevant field are followed or used.

As a specific example of the gene of the invention, there may bementioned the one deduced from the DNA sequence of a PCR product named“TSA305” which is to be shown later in the example section. Thenucleotide sequence thereof is as shown under SEQ ID NO:3.

Said gene is a human cDNA containing a coding region having thenucleotide sequence shown under SEQ ID NO:2 and coding for a novelpancreas-specific protein (hereinafter referred to as TSA305 protein)composed of 794 amino acid residues as shown under SEQ ID NO:1 and iscomposed of a total length of 7,885 nucleotides.

As a result of searching in the GenBank/EMBL database utilizing theFASTA program (Person, W. R., et al., Proc. Natl. Acad. Sci. USA, 85,2444-2448 (1988)), it was confirmed that the product of expression ofthe TSA305 gene of the invention, namely the TSA305 protein, has a veryhigh level of homology with the nematode sel-1 gene (cf. the referencecited above). In view of this fact, it is considered that the gene ofthe invention, like the above-mentioned sel-1, act suppressively onNotch/lin-12 which is a cancer-related gene considered to be involved inembryogenesis in general.

The locus of the gene of the invention is q24.3-q31.1 of the 14thchromosome where a gene causative of insulin-dependent diabetes mellitus(IDDM) is considered to exist. In view of this fact, it is stronglysuggested that the gene of the invention be related with diabetes.

It was further revealed that the product of expression of the gene ofthe invention is a protein containing a fibronectin type II collagenbinding domain. Such collagen binding site close to the N terminalsuggests involvement of the protein in fibrogenesis and, based on this,it is strongly suggested that the gene of the invention be involved infibrosis.

In addition, since all of the pancreatic carcinoma preparations testedshowed a failure of expression of the gene of the invention and the geneis expressed mainly in normal pancreases, it is suggested that the geneof the invention be potentially valuable in forecasting malignanttransformation.

Thus, information and means very useful in elucidating, understanding,diagnosing, preventing and treating various diseases such as mammarycancer, leukemia, fibrosis, diabetes and pancreatic carcinoma, inparticular pancreatic carcinoma, are given as a result of providing theTSA305 gene and the product of its expression according to the presentinvention. The gene of the invention can judiciously be used also indeveloping a novel drug inducing the expression of the gene of theinvention which is utilizable in the treatment of various diseases suchas mentioned above. Furthermore, detection of the expression of the geneof the invention or the product of its expression in an individualanimal or a specific tissue or detection of a mutation (deletion orpoint mutation) of said gene or abnormal expression thereof, forinstance, is considered to be utilizable adequately in elucidating ordiagnosing the above diseases.

The gene of the invention is specifically represented by a genecontaining a nucleotide sequence coding for a protein having the aminoacid sequence shown under SEQ ID NO:1 or a gene which is apolynucleotide containing the nucleotide sequence shown under SEQ IDNO:2. However, the gene of the invention is not particularly limited tothese but may be, for example, a gene leading to a certain modificationin the above specific amino acid sequence or a gene having a certainlevel of homology with the above specific nucleotide sequence.

Thus, the gene of the invention also includes a gene containing anucleotide sequence coding for a protein having an amino acid sequencederived from the amino acid sequence shown under SEQ ID NO:1 bydeletion, substitution or addition of one or a plurality of amino acidresidues and having the same activity as that of TSA305. The extent andsite(s) of “deletion, substitution or addition of an amino acid residueor residues” are not particularly restricted if the modified protein isa product of the same effect which has the same function as the proteinhaving the amino acid sequence shown under SEQ ID NO:1. The term“plurality” used above means 2 or more, normally several.

While the modification (mutation) or the like of the above amino acidsequence may occur naturally, for example by mutation orposttranslational modification, artificial modification is also possiblebased on a nature-derived gene (for example, a specific example of thegene of the present invention). The present invention covers allmodified genes having the above characteristic without reference to thecause and means, among others, of such modification or mutation.

As examples of the above artificial means, there may be mentionedsite-specific mutagenesis [Methods in Enzymology, 154: 350, 367-382(1987; ibid., 100: 468 (1983); Nucleic Acids Res., 12: 9441 (1984); ZokuSeikagaku Jikken Koza (Experiments in Biochemistry, second series) 1:“Idensi Kenkyuho (Methods in Gene Research) II”, edited by theBiochemical Society of Japan, p. 105 (1986)] and other geneticengineering techniques, means of chemical synthesis such as thephosphotriester method or phosphoamidite method [J. Am. Chem. Soc., 89:4801 (1967): ibid., 91: 3350 (1969); Science, 1: 178 (1968); TetrahedronLett., 22: 1859 (1981); ibid., 24: 245 (1983)], and combinationsthereof.

In a mode of embodiment of the gene of the present invention, there maybe mentioned a gene which is a polynucleotide containing the whole orpart of the nucleotide sequence shown under SEQ ID NO:3. The openreading frame (nucleotide sequence shown under SEQ ID NO:2) containingin this nucleotide sequence also serves as an example of combination ofcodons specifying respective amino acid residues in the above amino acidsequence (SEQ ID NO:1). The gene of the invention is not limited to thisbut can of course have a nucleotide sequence in which an arbitrarycombination of codons is selected. The selection of codons can be madein the conventional manner, for example the codon usage in the hostemployed, among others, can be taken into consideration [Nucleic AcidsRes., 9: 43 (1981)].

While the gene of the invention is represented in terms of single strandDNA nucleotide sequence, as shown, for example, under SEQ ID NO:2, theinvention of course includes a polynucleotide having a nucleotidesequence complementary to such nucleotide sequence, or a componentcomprising both of these as well. It is not limited to a DNA such as acDNA.

Furthermore, as mentioned above, the gene of the invention is notlimited to a polynucleotide containing the whole or part of thenucleotide sequence shown under SEQ ID NO:2, but includes genescomprising a nucleotide sequence having a certain level of homology withsaid nucleotide sequence as well. As such genes, there may be mentionedthose at least capable of hybridizing with a DNA comprising thenucleotide sequence shown under SEQ ID NO:2 under such stringentconditions as mentioned below and incapable of being released therefromeven by washing under certain conditions.

Thus, mention may be made, as an example, of a gene having a nucleotidesequence which hybridizes with a DNA having the nucleotide sequenceshown under SEQ ID NO:2 under conditions: at 65° C. overnight in 6×SSCor at 37° C. overnight in 4×SSC containing 50% formamide and is notreleased from said DNA under washing conditions: 30 minutes at 65° C.with 2×SSC. Here, “SSC” means standard saline citrate; 1×SSC =0.15 MNaCl, 0.015 M sodium citrate).

The gene of the present invention can be produced and recovered withease by general genetic engineering techniques [see, for example,Molecular Cloning, 2nd Ed., Cold Spring Harbor Lab. Press (1989); ZokuSeikagaku Jikken Koza (Experiments in Biochemistry, second series)“Idensi Kenkyuho (Methods in Gene Research) I, II and III”, edited bythe Biochemical Society of Japan (1986)] based on the information on thesequence of a typical example thereof.

Specifically, the production/recovery can be carried out by constructinga cDNA library in the conventional manner from an appropriate origin inwhich the gene of the invention is expressed and selecting a desiredclone therefrom using an appropriate probe or antibody specific to thegene of the invention [Proc. Natl. Acad. Sci. USA, 78: 6613 (1981);Science, 222: 778 (1983)].

As examples of the origin of cDNA in the above process, there may bementioned various cells and tissues in which the gene of the inventionis expressed, cultured cells derived therefrom and the like, inparticular the pancreatic tissue. Isolation of total RNA from these,isolation and purification of mRNA, obtainment of cDNA and cloningthereof, among others, can all be performed in the conventional manner.cDNA libraries are also commercially available and such cDNA libraries,for example various cDNA libraries commercially available from ClontechLab. Inc. can also be used in the practice of the present invention.

The method of screening the cDNA library for the gene of the inventionis not particularly restricted but may be a conventional one. Asspecific examples, there may be mentioned the method comprisingselecting the corresponding cDNA clone by immunological screening usinga specific antibody to the protein produced by the cDNA, plaquehybridization or colony hybridization using a probe selectively bindingto the desired DNA sequence, and combinations of these.

As examples of the probe to be used here, there may generally bementioned DNAs chemically synthesized based on the information on thenucleotide sequence of the gene of the invention, among others. Ofcourse, it is also possible to successfully utilize the gene of theinvention already obtained as such or fragments thereof.

The screening for the gene of the invention can also be made by theprotein interaction cloning procedure using the TSA305 protein in lieuof the above specific antibody and, further, the screening methodcomprising using, as a screening probe, a sense or antisense primerdesigned based on the information on the nucleotide sequence of the geneof the invention can also be employed.

In accordance with the present invention, the mRNA expression levels incells under different conditions or a plurality of different cell groupscan be directly compared and investigated by the differential displaytechnique (Liand, P., et al., Science, 257: 967-971 (1992)).

In obtaining the gene of the present invention, DNA/RNA amplification bythe PCR technique [Science, 230: 1350 (1985)] can judiciously beutilized. In particular, in cases where it is difficult to obtain thefull-length cDNA from a library, the RACE technique (rapid amplificationof cDNA ends; Jikken Igaku (Experimental Medicine), 12 (6): 35 (1994)),in particular the 5′-RACE technique [Proc. Natl. Acad. Sci. USA, 85:8998 (1988)], for instance, is judiciously employed. The primers to beused when such PCR technique is employed can be adequately designedbased on the information on the sequence of the gene of the invention asrevealed by the present invention and can be synthesized in theconventional manner.

The amplified DNA/RNA fragments can be isolated and purified in theconventional manner, as mentioned above, for example by gelelectrophoresis.

The gene of the invention or various DNA fragments obtained in the abovemanner can be sequenced in the conventional manner, for example by thedideoxy method [Proc. Natl. Acad. Sci. USA, 74: 5463 (1977)] or theMaxam-Gilbert method [Methods in Enzymology, 65: 499 (1980)] or, in asimple and easy manner, by using a commercial sequencing kit or thelike.

By utilizing the gene of the present invention, it is possible toreadily produce the corresponding gene product stably in large amountsby using general genetic engineering techniques. Therefore, the presentinvention also provides a vector (expression vector) containing theTSA305 gene of the invention, host cells transformed with said vectorand a method of producing the TSA305 protein which comprises cultivatingsaid host cells.

The production method can be carried out according to the ordinaryrecombinant DNA technology [see, for example, Science, 2: 1431 (1984);Biochem. Biophys. Res. Comm., 130: 692 (1985); Proc. Natl. Acad. Sci.USA, 80: 5990 (1983); and the references cited above].

Both prokaryotes and eukaryotes can be used as the host cells mentionedabove. As prokaryotic hosts, there may be mentioned a wide variety ofones in general use, such as Escherichia coli, Bacillus subtilis, etc.,and preferred examples are those included among Escherichia colistrains, in particular the Escherichia coli K 12 strain. The eukaryotichost cells include vertebrate cells and yeast cells, among others. Asthe former, COS cells [Cell, 23: 175 (1981)], which are simian cells,chinese hamster ovary cells and the dihydrofolate reductase-deficientstrain thereof [Proc. Natl. Acad. Sci. USA, 77: 4216 (1980)], forinstance, are judiciously used and, as the latter, yeast cells belongingto the genus Saccharomyces and the like are judiciously used. Of course,the host cells are not limited to these.

Where prokaryotic cells are used as the host, an expression plasmid canjudiciously be used which is constructed using a vector capable ofreplicating in said host cells and providing this vector with a promoterand the SD (Shine and Dalgarno) sequence upstream of the gene of theinvention and further with an initiation codon (e.g. ATG) necessary forthe initiation of protein synthesis so that the gene of the inventionmay be expressed. Often used as the above vector are generallyEscherichia coli-derived plasmids, for example pBR322, pBR325, pUC12 andpUC13. The vector is not limited to these, however, but various knownvectors may be utilized. As commercially available vectors of the abovekind which can be used in expression systems in which Escherichia coliis used, there may be mentioned, for example, pGEX-4T (AmershamPharmacia Biotech), pMAL-C2, pMAl-P2 (New Englans Biolabs), pET21,pET21/lacq (Invitrogen) and pBAD/His (Invitrogen).

As the expression vector in the case of vertebrate cells being used asthe host, there may be mentioned one generally having a promoter locatedupstream of the gene of the invention which is to be expressed, an RNAsplicing site, a polyadenylation site and transcription terminationsequence. When necessary, this may further have an origin ofreplication. As specific examples of said expression vector, there maybe mentioned pSV2dhfr having the early promoter of SV40 [Mol. Cell.Biol., 1: 854 (1981)] and the like. In addition to the above, variousknown commercial vectors can also be used. As commercial vectors of suchkind which are to be utilized in expression systems in which animalcells are used, there may be mentioned, among others, vectors for animalcells, such as pEGFP-N, pEGFP-C (Clontech), pIND (Invitrogen) andpcDNA3.1/His (Invitrogen), and vectors for insect cells, such aspFastBac HT (Gibco BRL), pAcGHLT (PharMingen), pAc5/V5-His, pMT/V5-Hisand pMT/Bip/V5-His (the latter three: Invitrogen).

As specific examples of the expression vector to be used when yeastcells are used as the host, there may be mentioned, among others, pAM82having a promoter for the acid phosphatase gene [Proc. Natl. Acad. Sci.USA, 80; 1 (1983)] and the like. Commercial expression vectors for yeastcells include, among others, pPICZ (Invitrogen) and pPICZα(Invitrogen).

The promoter is not particularly restricted, either. When an Escherichiaspecies is used as the host, the tryptophan (trp) promoter, lpppromoter, lac promoter, recA promoter, PL/PR promoter or the like canjudiciously be utilized. When the host is a Bacillus species, the SP01promoter, SP02 promoter, penP promoter or the like is preferred. As forthe promoter to be used when a yeast species is the host, the pH05promoter, PGK promoter, GAP promoter or ADH promoter, for instance, canjudiciously be used. As preferred examples of the promoter to be usedwhen animal cells are used as the host, there may be mentionedSV40-derived promoters, retrovirus promoters, and the metallothioneinpromoter, heat shock promoter, cytomegalovirus promoter and SRα promter.

Conventional fused protein expression vectors can also judiciously beused as the expression vector for the gene of the present invention. Asspecific examples of such vectors, there may be mentioned pGEX (Promega)for the expression of a protein fused with glutathione-S-transferase(GST) and the like.

The method of introducing the desired recombinant DNA (expressionvector) into host cells for transforming the same is not particularlyrestricted, either, but various general methods can be employed. Thetransformant obtained can be cultivated in the conventional manner,whereby the desired TSA305 protein encoded by the gene of the presentinvention is expressed/produced and accumulated or secreted within oroutside the transformant cells or on the cell membrane.

The medium to be used in the above cultivation can adequately beselected from among various conventional ones according to the hostcells employed, and the cultivation can be conducted under conditionssuited for the growth of the host cells.

The thus-obtained recombinant protein (TSA305 protein) can be isolatedand purified, as desired, by various separation procedures utilizing itsphysical and/or chemical properties, among others [see, for example,“Seikagaku (Biochemical) Data Book II”, pages 1175-1259, 1st edition,1st printing, published Jun. 23, 1980 by Tokyo Kagaku Dojin;Biochemistry, 25 (25); 8274 (1986); and Eur. J. Biochem., 163: 313(1987)]. As said methods, there may specifically be mentioned, forexample, ordinary reconstitution treatment, treatment with a proteinprecipitating agent (salting out), centrifugation, osmotic shockprocedure, sonication, ultrafiltration, various chromatographictechniques such as molecular sieve chromatography (gel filtration),adsorption chromatography, ion exchange chromatography, affinitychromatography and high-performance liquid chromatography (HPLC),dialysis and combinations of these. Particularly preferred among theabove methods is affinity chromatography using a column to which aspecific antibody to the TSA305 protein of the invention is bound.

Thus, the present invention further provides the novel TSA305 proteinitself as obtained, for example, in the above manner. As mentionedhereinabove, said protein has a high level of homology with the nematodesel-1 and can produce an inhibitory effect on various kinds of cancerand therefore useful in the pharmaceutical field.

This TSA305 protein can also be utilized as an immunogen for producingan antibody specific to said protein. The component to be used here asthe antigen may be the protein mass-produced by the genetic engineeringtechniques mentioned above or a fragment thereof, for instance. Byutilizing such antigen, it is possible to obtain the desired antiserum(polyclonal antibody) or monoclonal antibody. The methods of producingsaid antibody are themselves well known to those skilled in the art and,in the practice of the present invention as well, these conventionalmethods can be followed [see, for example, Zoku Seikagaku Jikken Koza(Experiments in Biochemistry, second series) “Men-eki Seikagaku kenkyuho(Methods in Immunobiochemistry)”, edited by the Biochemical Society ofJapan (1986)].

Thus, for example, the animal to be immunized for obtaining antisera canbe arbitrarily selected from among ordinary animals such as rabbits,guinea pigs, rats, mice and chickens, and immunization with the antigenmentioned above, blood collection and other procedures can also becarried out in the conventional manner.

The monoclonal antibody, too, can be produced in the conventional mannerby producing hybrid cells from plasmocytes (immunocytes) of an animalimmunized with the immunogen mentioned above and plasmacytoma cells,selecting a desired antibody-producing clone from among them, andcultivating said clone. The animal to be immunized is generally selectedtaking into consideration the compatibility with the plasmacytoma cellsemployed for cell fusion and, generally, mice or rats, among others, areadvantageously used. The immunization can be conducted in the samemanner as in the above-mentioned case of antisera and, if desirable, anordinary adjuvant or the like may be used in combination.

The plasmacytoma cells to be used for cell fusion are not particularlyrestricted but, for example, various myeloma cells such as p3(p3/x63-Ag8) [Nature, 256: 495-497 (1975)], p3-U1 [Current Topics inMicrobiology and Immunology, 81: 1-7 (1978)], NS-1 [Eur. J. Immmunol.,6: 511-519 (1976)], MPC-11 [Cell, 8: 405-415 (1976)], SP2/0 [Nature,276: 269-271 (1978) and the like, R210 in rats [Nature, 211: 131-133(1979)] and the like as well as cells derived therefrom all can be used.

The fusion of the above immunocytes and plasmacytoma cells can beperformed by a known method in the presence of a conventional fusionaccelerator such as polyethylene glycol (PEG) or Sendai virus (HVJ) andthe desired hybridomas can also be isolated in the conventional manner[e.g. Meth. in Enzymol., 73: 3 (1981); Zoku Seikagaku Jikken Koza(Experiments in Biochemistry, second series) cited above].

The desired antibody-producing cell line can be searched for and amonoclonal antibody can be derived therefrom in the conventional manner.Thus, for example, the search for an antibody-producing cell line can becarried out using the above-mentioned antigen of the present inventionby various methods generally used in detecting antibodies, such as theELISA technique [Meth. in Enzymol., 70: 419-439 (1980)], plaquetechnique, spot technique, agglutination reaction technique, Ouchterlonytechnique, and radioimmunoassay.

The antibody of the invention can be collected from the thus-obtainedhybridomas, for example, by cultivating said hybridomas in theconventional manner and collecting the culture supernatant or byadministering the hybridomas to an mammal compatible therewith and,after hybridoma growth, collecting the ascitic fluid. The former methodis suited for obtaining a high-purity antibody while the latter methodis suited for mass production of an antibody. The thus-obtained antibodycan further be purified by conventional means such as salting out, gelfiltration and affinity chromatography.

The thus-obtained antibody is characterized by its ability to bind tothe TSA305 protein of the invention and can advantageously be utilizedin the above-mentioned purification of the TSA305 protein and inassaying or discriminating the same by immunological techniques. Thepresent invention thus provides such novel antibody as well.

Further, based on the information on the sequence of the gene of theinvention as revealed by the present invention, the expression of thegene of the invention in individuals or in various tissues can bedetected, for example by utilizing the whole or part of the nucleotidesequence of said gene.

Such detection can be carried out in the conventional manner, forexample by RNA amplification by RT-PCR [reverse transcribed polymerasechain reaction; E. S. Kawasaki et al., Amplification of RNA. In PCRProtocol, A Guide to methods and applications, Academic Press, Inc., SanDiego, 21-27 (1991)], northern blot analysis [Molecular Cloning, ColdSpring Harbor Lab. (1989)], in situ RT-PCR [Nucl. Acids Res., 21:3159-3166 (1993)], in situ hybridization or a like technique forassaying the same on the cellular level or by the NASBA technique[nucleic acid sequence-based amplification; Nature, 350: 91-92 (1991)]or other various techniques. All can give good results.

When the RT-PCR technique is employed, the primers to be used are notlimited in any way provided that they are specific to the gene of theinvention and enable specific amplification of said gene alone. Thesequences thereof can be adequately designed based on the geneticinformation according to the present invention. Generally, each may havea partial sequence comprising about 20 to 30 nucleotides.

In this way, the present invention provides DNA fragments useful asspecific primers and/or specific probes in detecting the TSA305 geneaccording to the invention as well.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a photograph, in lieu of a drawing, illustrating thedistribution of the gene of the invention in human tissues as examinedby the northern blot analysis described in Example 1 under (2).

FIG. 2 is a photograph, in lieu of a drawing, illustrating the resultsof RT-PCR analysis of normal pancreatic cells and four cell lines asobtained in Example 1 (4). The results for TSA305 are shown in the uppersection and the results for β₂-microglobulin as a control are shown inthe lower section.

FIG. 3 is a photograph, in lieu of a drawing, illustrating the resultsof RT-PCR analysis of pancreatic carcinoma samples and others asobtained in Example 1 (5). The results for TSA305 are shown in the uppersection and the results for β₂-microglobulin as a control are shown inthe lower section.

BEST MODE FOR CARRYING OUT THE INVENTION

The following examples are given for illustrating the present inventionin more detail.

EXAMPLE 1

(1-1) Method of Manifestation by Labeling with [α-³³P]ATP

For identifying the human gene expressed in a tissue-specific process,the method of manifestation by labeling with [α-³³P]ATP was used. Theprocedure of said method was followed essentially according to themethod of Liang (Liang, P., et al., Science, 257: 967-971 (1992)), asmentioned below.

Thus, polyA RNA (0.2 μg) isolated from each of 13 human tissues (adultbrain, fetal brain, lung, liver, stomach, pancreas, spleen, mammarygland, bladder, placenta, testis, kidney and heart; products ofClontech) was mixed with 25 pmol of 3′-anchored oligo-dT primer G(T)15MA(M being a mixture of G, A and C) in 8 μl of diethylpyrocarbonate-treated water and the mixture was heated at 65° C. for 5minutes. To this solution were added 4 μl of 5× First strand buffer(product of BRL), 2 μl of 0.1 M DTT (product of BRL), 1 μl of 250 mMdNTPs (product of BRL), 1 μl of ribonuclease inhibitor (40 units;product of Toyobo) and 1 μl of SuperScript II reverse transcriptase (200units; product of BRL). The final volume of each reaction mixture was 20μl. Each solution was incubated at 37° C. for 1 hour and then 2.5-folddiluted by addition of 30 μl of distilled water and the dilution wasstored at −20° C. until the time of use.

cDNA was amplified by PCR in the presence of [α-³³P]ATP-labeled (productof Amersham) 3′-anchored primer. This cDNA amplification by PCR wasconducted in the following manner. Thus, 20 μl of each PCR mixturecontained 2 μl of RT reaction mixture, 2 μl of 10 ×PCR buffer (productof Takara), 4 μl of 2.5 mM dNTPs, 0.25 μl of ExTaq DNA polymerase (5units/ml; product of Takara), 25 pmol of [Δ-³³P]ATP-labeled 3′-anchoredoligo-dT primer and 25 pmol of 5′-primer (No. 20, decamerdeoxyoligo-nucleotide primer having an arbitrary sequence, in this casethe nucleotide sequence shown under SEQ ID NO:4). The PCR reaction wascarried out under the following conditions. Thus, one cycle wasconducted at 95° C. for 3 minutes, at 40° C. for 5 minutes and at 72° C.for 5 minutes, then 40 cycles were conducted each at 95° C. for 0.5minutes, at 40° C. for 2 minutes and at 72° C. for 1 minute and,finally, the reaction was allowed to proceed at 72° C. for 5 minutes.

Each PCR reaction sample was extracted with ethanol and resuspended informamide-sequencing dye and the reaction was allowed to proceed on a 6%acrylamide-7.5 M ureas sequencing gel. The gel was dried withoutfixation and autoradiography was carried out overnight.

(1-2) Subcloning of the Amplified cDNA Fragment

3MM filter paper with the dried gel placed thereon was marked withradioactive ink in advance. By checking the autoradiogram against thismark, the gel containing the desired cDNA-containing band was excisedtogether with the 3MM filter paper and stirred with 300 μl of dH₂O for 1hour. After removal of the polyacrylamide gel and filter paper, the cDNAwas rerecovered by ethanol precipitation in the presence of 1 μl of 10mg/ml glycogen and 0.3 M NaOAc as a carrier and redissolved in 10 μl ofdH₂O. For reamplification, 5 μl of this solution was used. The PCRconditions and primers were the same as those in the first PCR. Thereamplification product having an appropriate size was recovered as thefirst PCR product, and the PCR product was then cloned into the pUC118vector (product of Takara) at the HincII site. The nucleotide sequencewas determined using an ABI 377 automated sequencer (product of AppliedBiosystems).

The different patterns manifested upon use of the mRNAs isolated fromthe 13 human tissues were compared and, as a result, a PCR productspecifically expressed in pancreas was identified. This was namedTSA305.

This product was composed of 371 nucleotides. Comparison of the data onthis nucleotide with the DNA sequences occurring in the GenBank/EMBLdata base using the FASTA program (Person, W. R., et al., Proc. Natl.Acad. Sci. USA, 85: 2444-2448 (1988)) revealed that this PCR product hasno homology with any of other known DNA sequences.

(1-3) cDNA Screening

A human normal pancreas cDNA library was constructed usingoligo(dT)+random hexamer-primed human normal pancreas cDNA and Uni-ZAP™XR (product of Stratagene). The total of 1×10₆ clones were isolated bythe method mentioned above and subjected to screening using a [Δ-³³P]-dCTP-labeled cDNA fragment. Positive clones were selected and theinsert cDNA portions thereof were excised in vivo in pBluescript IISK(−).

As a result, about 100 plaques were identified as corresponding toTSA305. Based on this result, the percent transcription among all RNAswas calculated to be about 0.01%. The assembled cDNA sequence (TSA305)homologous with TSA305 comprises 7,885 nucleotides containing an openreading frame of 2,382 nucleotides coding for a protein composed of 794amino acid residues with a calculated molecular weight of 88,768 Da.

Based on the primary sequence, it was revealed that this gene product(TSA305 protein) is a protein containing a fibronectin type II collagenbinding domain.

The locus thereof was found to be q24.3-q31.1 on the 14th chromosomewhere a gene causative of insulin-dependent diabetes mellitus (IDDM) isconsidered to exist.

The TSA305 gene of the present invention showed a high level of homologywith the nematode sel-1.

(2) Expression in Tissues

For checking the expression profiles of TSA305 in tissues, northern blotanalysis was carried out using various human tissues.

For the northern blot analysis, human MTN (Multiple Tissue Northern)blots I and II (products of Clontech) were used. The cDNA fragments werelabeled with [α-³³P]-dCTP by PCR using a set of primers with T3 and T7promoter sequences. The amplification product-containing membrane wasprehybridized (the conditions were as indicated in the productprotocol), followed by hybridization according to the product protocol.

After hybridization, the membrane was washed and exposed to anautoradiograph at −80° C. for 24 hours. The results are shown in FIG. 1.

In the figure, the human tissues used were heart, brain, placenta, lung,liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate,testis, ovary, small intestine, colon and peripheral blood leukocytes.

In the figure, a transcript homologous with TSA305 was observedspecifically in the pancreas.

(3) FISH

FISH was carried out for chromosome arrangement according to a knownmethod (Takahashi, E., et al., Hum. Genet., 86: 14-16 (1990)) using 0.5μg of each cosmide DNA as a probe. FISH was detected with a Provia 100film (product of Fuji, ISO 100) or a CCD camera system (Applied Imaging,product of Sightvision).

As a result, the signals obtained by testing 100 typical cells at(pro)metaphase by R banding were found localized on the bandsq24.3-q31.1 of the 14th chromosome. Therefore, the locus of localizationof the TSA305 on the chromosome could be identified as 14q24.3-q31.1.

(4) Expression of Transcript in Pancreatic Carcinoma Cell Lines and inPancreatic Carcinoma Tissues as Revealed by RT-PCR Analysis

To check whether the expression of the TSA305 gene varies in humanpancreatic carcinoma cell lines and pancreatic carcinoma tissues, fourcell lines (Aspc1 (metastatic adenocarcinoma; J. Natl. Cancer Inst., 67:563-569 (1981)), Bxpc3 (adenocarcinoma, undifferentiated; CancerInvest., 4: 15-23 (1986)), MiaPaca2 (adenocarcinoma; Int. J. Cancer, 19:128-135 (1977)) and PANC1 (epithelioid, pancreatic duct carcinoma; Int.J. Cancer, 15: 741-747 (1975)) and 9 pancreatic carcinoma tissues (giftsfrom Dr. Nakamura at the University of Tokyo Institute of MedicalSciences) were subjected to RT-PCR analysis.

Thus, 10 μl of the total RNA isolated from each cell line or pancreaticcarcinoma tissue using ISOGEN (product of Wako) was treated with 10units of RNase-free DNase I (product of Boehringer Mannheim) for 15minutes, followed by two repetitions of extraction withphenol-chloroform and precipitation with ethanol. The single-strandedcDNA was synthesized using Superscript I™ RNase H reverse transcriptase(product of Life Technology) with oligo-d(T) and random primers. A 2-μlportion of each product was used for PCR amplification.

The primers P1 and P2S having the nucleotide sequences shown under SEQID NO:5 and SEQ ID NO:6, respectively, were used in 25 cycles of PCRamplification.

The PCR reaction was carried out in 20 μl of a solution containing 25 ngof cDNA, 10 μM each primer, 2.5 mM dNTP and 0.25 U of Extaq DNApolymerase (product of Takara). Each PCR product was dissolved in 1.5%agarose gel stained with ethidium bromide.

The four cell lines (lane 1=Aspc1; lane 2 =Bxpc3; lane 3=MiaPaca2; lane4=PANC1) and a normal pancreatic tissue (normal pancreas, lane 5) wereanalyzed by RT-PCR in the above manner. The results are as shown in FIG.2. The results for TSA305 are shown in the upper section and the resultsfor μ₂-microglobulin as a control are shown in the lower section.

From the figure, it was found that the expression of TSA305 is notdetected in any of cancer tissues but is detected only in normalpancreatic tissues (cf. lane 5).

(5) Expression of the TSA305 Gene in Pancreatic Carcinoma (RT-PCR)

The expression of the TSA305 gene was checked in pancreatic carcinomapatient-derived samples (1T, 2T, 3T, 5T, 6T, 7T, 10T and 11T),pancreatic carcinoma (Tumor Pancreas) and normal pancreas (Invitrogen;Human Normal Pancreas) as well as a cancerous portion (23T) and anoncancerous portion (23N) of the same patient by the RT-PCR technique,as follows.

mRNA was extracted from each sample and the segment of 1581-2382 bp (801base pairs) of TSA305 was amplified by RT-PCR and tested for detectingexpression or no expression. As a concentration control,β₂-microglobulin was used. The results are shown in FIG. 3.

From the figure, reduced expression or lack of expression of the TSA305gene was observed in all pancreatic carcinoma samples as compared withthe normal pancreas.

INDUSTRIAL APPLICABILITY

According to the present invention, the novel pancreas-specific TSA305gene and the protein encoded thereby are provided and, by utilizingthese, a technology useful, among others, in elucidating, diagnosing,preventing and treating cancers, such as pancreatic carcinoma, ormalignant transformation is provided.

SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 6 <210> SEQ ID NO 1 <211>LENGTH: 794 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE:<221> NAME/KEY: MISC_FEATURE <223> OTHER INFORMATION: Human NormalPancreas cDNA Library <400> SEQUENCE: 1 Met Arg Val Arg Ile Gly Leu ThrLeu Leu Leu Cys Ala Val Leu Leu 1 5 10 15 Ser Leu Ala Ser Ala Ser SerAsp Glu Glu Gly Ser Gln Asp Glu Ser 20 25 30 Leu Asp Ser Lys Thr Thr LeuThr Ser Asp Glu Ser Val Lys Asp His 35 40 45 Thr Thr Ala Gly Arg Val ValAla Gly Gln Ile Phe Leu Asp Ser Glu 50 55 60 Glu Ser Glu Leu Glu Ser SerIle Gln Glu Glu Glu Asp Ser Leu Lys 65 70 75 80 Ser Gln Glu Gly Glu SerVal Thr Glu Asp Ile Ser Phe Leu Glu Ser 85 90 95 Pro Asn Pro Glu Asn LysAsp Tyr Glu Glu Pro Lys Lys Val Arg Lys 100 105 110 Pro Ala Leu Thr AlaIle Glu Gly Thr Ala His Gly Glu Pro Cys His 115 120 125 Phe Pro Phe LeuPhe Leu Asp Lys Glu Tyr Asp Glu Cys Thr Ser Asp 130 135 140 Gly Arg GluAsp Gly Arg Leu Trp Cys Ala Thr Thr Tyr Asp Tyr Lys 145 150 155 160 AlaAsp Glu Lys Trp Gly Phe Cys Glu Thr Glu Glu Glu Ala Ala Lys 165 170 175Arg Arg Gln Met Gln Glu Ala Glu Met Met Tyr Gln Thr Gly Met Lys 180 185190 Ile Leu Asn Gly Ser Asn Lys Lys Ser Gln Lys Arg Glu Ala Tyr Arg 195200 205 Tyr Leu Gln Lys Ala Ala Ser Met Asn His Thr Lys Ala Leu Glu Arg210 215 220 Val Ser Tyr Ala Leu Leu Phe Gly Asp Tyr Leu Pro Gln Asn IleGln 225 230 235 240 Ala Ala Arg Glu Met Phe Glu Lys Leu Thr Glu Glu GlySer Pro Lys 245 250 255 Gly Gln Thr Ala Leu Gly Phe Leu Tyr Ala Ser GlyLeu Gly Val Asn 260 265 270 Ser Ser Gln Ala Lys Ala Leu Val Tyr Tyr ThrPhe Gly Ala Leu Gly 275 280 285 Gly Asn Leu Ile Ala His Met Val Leu GlyTyr Arg Tyr Trp Ala Gly 290 295 300 Ile Gly Val Leu Gln Ser Cys Glu SerAla Leu Thr His Tyr Arg Leu 305 310 315 320 Val Ala Asn His Val Ala SerAsp Ile Ser Leu Thr Gly Gly Ser Val 325 330 335 Val Gln Arg Ile Arg LeuPro Asp Glu Val Glu Asn Pro Gly Met Asn 340 345 350 Ser Gly Met Leu GluGlu Asp Leu Ile Gln Tyr Tyr Gln Phe Leu Ala 355 360 365 Glu Lys Gly AspVal Gln Ala Gln Val Gly Leu Gly Gln Leu His Leu 370 375 380 His Gly GlyArg Gly Val Glu Gln Asn His Gln Arg Ala Phe Asp Tyr 385 390 395 400 PheAsn Leu Ala Ala Asn Ala Gly Asn Ser His Ala Met Ala Phe Leu 405 410 415Gly Lys Met Tyr Ser Glu Gly Ser Asp Ile Val Pro Gln Ser Asn Glu 420 425430 Thr Ala Leu His Tyr Phe Lys Lys Ala Ala Asp Met Gly Asn Pro Val 435440 445 Gly Gln Ser Gly Leu Gly Met Ala Tyr Leu Tyr Gly Arg Gly Val Gln450 455 460 Val Asn Tyr Asp Leu Ala Leu Lys Tyr Phe Gln Lys Ala Ala GluGln 465 470 475 480 Gly Trp Val Asp Gly Gln Leu Gln Leu Gly Ser Met TyrTyr Asn Gly 485 490 495 Ile Gly Val Lys Arg Asp Tyr Lys Gln Ala Leu LysTyr Phe Asn Leu 500 505 510 Ala Ser Gln Gly Gly His Ile Leu Ala Phe TyrAsn Leu Ala Gln Met 515 520 525 His Ala Ser Gly Thr Gly Val Met Arg SerCys His Thr Ala Val Glu 530 535 540 Leu Phe Lys Asn Val Cys Glu Arg GlyArg Trp Ser Glu Arg Leu Met 545 550 555 560 Thr Ala Tyr Asn Ser Tyr LysAsp Gly Asp Tyr Asn Ala Ala Val Ile 565 570 575 Gln Tyr Leu Leu Leu AlaGlu Gln Gly Tyr Glu Val Ala Gln Ser Asn 580 585 590 Ala Ala Phe Ile LeuAsp Gln Arg Glu Ala Ser Ile Val Gly Glu Asn 595 600 605 Glu Thr Tyr ProArg Ala Leu Leu His Trp Asn Arg Ala Ala Ser Gln 610 615 620 Gly Tyr ThrVal Ala Arg Ile Lys Leu Gly Asp Tyr His Phe Tyr Gly 625 630 635 640 PheGly Thr Asp Val Asp Tyr Glu Thr Ala Phe Ile His Tyr Arg Leu 645 650 655Ala Ser Glu Gln Gln His Ser Ala Gln Ala Met Phe Asn Leu Gly Tyr 660 665670 Met His Glu Lys Gly Leu Gly Ile Lys Gln Asp Ile His Leu Ala Lys 675680 685 Arg Phe Tyr Asp Met Ala Ala Glu Ala Ser Pro Asp Ala Gln Val Pro690 695 700 Val Phe Leu Ala Leu Cys Lys Leu Gly Val Val Tyr Phe Leu GlnTyr 705 710 715 720 Ile Arg Glu Thr Asn Ile Arg Asp Met Phe Thr Gln LeuAsp Met Asp 725 730 735 Gln Leu Leu Gly Pro Glu Trp Asp Leu Tyr Leu MetThr Ile Ile Ala 740 745 750 Leu Leu Leu Gly Thr Val Ile Ala Tyr Arg GlnArg Gln His Gln Asp 755 760 765 Met Pro Ala Pro Arg Pro Pro Gly Pro ArgPro Ala Pro Pro Gln Gln 770 775 780 Glu Gly Pro Pro Glu Gln Gln Pro ProGln 785 790 <210> SEQ ID NO 2 <211> LENGTH: 2382 <212> TYPE: DNA <213>ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <223>OTHER INFORMATION: Human Normal Pancreas cDNA Library <400> SEQUENCE: 2atgcgggtcc ggatagggct gacgctgctg ctgtgtgcgg tgctgctgag cttggcctcg 60gcgtcctcgg atgaagaagg cagccaggat gaatccttag attccaagac tactttgaca 120tcagatgagt cagtaaagga ccatactact gcaggcagag tagttgctgg tcaaatattt 180cttgattcag aagaatctga attagaatcc tctattcaag aagaggaaga cagcctcaag 240agccaagagg gggaaagtgt cacagaagat atcagctttc tagagtctcc aaatccagaa 300aacaaggact atgaagagcc aaagaaagta cggaaaccag ctttgaccgc cattgaaggc 360acagcacatg gggagccctg ccacttccct tttcttttcc tagataagga gtatgatgaa 420tgtacatcag atgggaggga agatggcaga ctgtggtgtg ctacaaccta tgactacaaa 480gcagatgaaa agtggggctt ttgtgaaact gaagaagagg ctgctaagag acggcagatg 540caggaagcag aaatgatgta tcaaactgga atgaaaatcc ttaatggaag caataagaaa 600agccaaaaaa gagaagcata tcggtatctc caaaaggcag caagcatgaa ccataccaaa 660gccctggaga gagtgtcata tgctctttta tttggtgatt acttgccaca gaatatccag 720gcagcgagag agatgtttga gaagctgact gaggaaggct ctcccaaggg acagactgct 780cttggctttc tgtatgcctc tggacttggt gttaattcaa gtcaggcaaa ggctcttgta 840tattatacat ttggagctct tgggggcaat ctaatagccc acatggtttt gggttacaga 900tactgggctg gcatcggcgt cctccagagt tgtgaatctg ccctgactca ctatcgtctt 960gttgccaatc atgttgctag tgatatctcg ctaacaggag gctcagtagt acagagaata 1020cggctgcctg atgaagtgga aaatccagga atgaacagtg gaatgctaga agaagatttg 1080attcaatatt accagttcct agctgaaaaa ggtgatgtac aagcacaggt tggtcttgga 1140caactgcacc tgcacggagg gcgtggagta gaacagaatc atcagagagc atttgactac 1200ttcaatttag cagcaaatgc tggcaattca catgccatgg cctttttggg aaagatgtat 1260tcggaaggaa gtgacattgt acctcagagt aatgagacag ctctccacta ctttaagaaa 1320gctgctgaca tgggcaaccc agttggacag agtgggcttg gaatggccta cctctatggg 1380agaggagttc aagttaatta tgatctagcc cttaagtatt tccagaaagc tgctgaacaa 1440ggctgggtgg atgggcagct acagcttggt tccatgtact ataatggcat tggagtcaag 1500agagattata aacaggcctt gaagtatttt aatttagctt ctcagggagg ccatatcttg 1560gctttctata acctagctca gatgcatgcc agtggcaccg gcgtgatgcg atcatgtcac 1620actgcagtgg agttgtttaa gaatgtatgt gaacgaggcc gttggtctga aaggcttatg 1680actgcctata acagctataa agatggcgat tacaatgctg cagtgatcca gtacctcctc 1740ctggctgaac agggctatga agtggcacaa agcaatgcag cctttattct tgatcagaga 1800gaagcaagca ttgtaggtga gaatgaaact tatcccagag ctttgctaca ttggaacagg 1860gccgcctctc aaggctatac tgtggctaga attaagctcg gagactacca tttctatggg 1920tttggcaccg atgtagatta tgaaactgca tttattcatt accgtctggc ttctgagcag 1980caacacagtg cacaagctat gtttaatctg ggatatatgc atgagaaagg actgggcatt 2040aaacaggata ttcaccttgc gaaacgtttt tatgacatgg cagctgaagc cagcccagat 2100gcacaagttc cagtcttcct agccctctgc aaattgggcg tcgtctattt cttgcagtac 2160atacgggaaa caaacattcg agatatgttc acccaacttg atatggacca gcttttggga 2220cctgagtggg acctttacct catgaccatc attgcgctgc tgttgggaac agtcatagct 2280tacaggcaaa ggcagcacca agacatgcct gcacccaggc ctccagggcc acggccagct 2340ccaccccagc aggaggggcc accagagcag cagccaccac ag 2382 <210> SEQ ID NO 3<211> LENGTH: 7885 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220>FEATURE: <221> NAME/KEY: exon <222> LOCATION: (46)..(2427) <221>NAME/KEY: misc_feature <223> OTHER INFORMATION: Human Normal PancreascDNA Library <400> SEQUENCE: 3 gcgaaggcga cagctctagg ggttggcaccggccccgaga ggagg atg cgg gtc cgg 57 Met Arg Val Arg 1 ata ggg ctg acgctg ctg ctg tgt gcg gtg ctg ctg agc ttg gcc tcg 105 Ile Gly Leu Thr LeuLeu Leu Cys Ala Val Leu Leu Ser Leu Ala Ser 5 10 15 20 gcg tcc tcg gatgaa gaa ggc agc cag gat gaa tcc tta gat tcc aag 153 Ala Ser Ser Asp GluGlu Gly Ser Gln Asp Glu Ser Leu Asp Ser Lys 25 30 35 act act ttg aca tcagat gag tca gta aag gac cat act act gca ggc 201 Thr Thr Leu Thr Ser AspGlu Ser Val Lys Asp His Thr Thr Ala Gly 40 45 50 aga gta gtt gct ggt caaata ttt ctt gat tca gaa gaa tct gaa tta 249 Arg Val Val Ala Gly Gln IlePhe Leu Asp Ser Glu Glu Ser Glu Leu 55 60 65 gaa tcc tct att caa gaa gaggaa gac agc ctc aag agc caa gag ggg 297 Glu Ser Ser Ile Gln Glu Glu GluAsp Ser Leu Lys Ser Gln Glu Gly 70 75 80 gaa agt gtc aca gaa gat atc agcttt cta gag tct cca aat cca gaa 345 Glu Ser Val Thr Glu Asp Ile Ser PheLeu Glu Ser Pro Asn Pro Glu 85 90 95 100 aac aag gac tat gaa gag cca aagaaa gta cgg aaa cca gct ttg acc 393 Asn Lys Asp Tyr Glu Glu Pro Lys LysVal Arg Lys Pro Ala Leu Thr 105 110 115 gcc att gaa ggc aca gca cat ggggag ccc tgc cac ttc cct ttt ctt 441 Ala Ile Glu Gly Thr Ala His Gly GluPro Cys His Phe Pro Phe Leu 120 125 130 ttc cta gat aag gag tat gat gaatgt aca tca gat ggg agg gaa gat 489 Phe Leu Asp Lys Glu Tyr Asp Glu CysThr Ser Asp Gly Arg Glu Asp 135 140 145 ggc aga ctg tgg tgt gct aca acctat gac tac aaa gca gat gaa aag 537 Gly Arg Leu Trp Cys Ala Thr Thr TyrAsp Tyr Lys Ala Asp Glu Lys 150 155 160 tgg ggc ttt tgt gaa act gaa gaagag gct gct aag aga cgg cag atg 585 Trp Gly Phe Cys Glu Thr Glu Glu GluAla Ala Lys Arg Arg Gln Met 165 170 175 180 cag gaa gca gaa atg atg tatcaa act gga atg aaa atc ctt aat gga 633 Gln Glu Ala Glu Met Met Tyr GlnThr Gly Met Lys Ile Leu Asn Gly 185 190 195 agc aat aag aaa agc caa aaaaga gaa gca tat cgg tat ctc caa aag 681 Ser Asn Lys Lys Ser Gln Lys ArgGlu Ala Tyr Arg Tyr Leu Gln Lys 200 205 210 gca gca agc atg aac cat accaaa gcc ctg gag aga gtg tca tat gct 729 Ala Ala Ser Met Asn His Thr LysAla Leu Glu Arg Val Ser Tyr Ala 215 220 225 ctt tta ttt ggt gat tac ttgcca cag aat atc cag gca gcg aga gag 777 Leu Leu Phe Gly Asp Tyr Leu ProGln Asn Ile Gln Ala Ala Arg Glu 230 235 240 atg ttt gag aag ctg act gaggaa ggc tct ccc aag gga cag act gct 825 Met Phe Glu Lys Leu Thr Glu GluGly Ser Pro Lys Gly Gln Thr Ala 245 250 255 260 ctt ggc ttt ctg tat gcctct gga ctt ggt gtt aat tca agt cag gca 873 Leu Gly Phe Leu Tyr Ala SerGly Leu Gly Val Asn Ser Ser Gln Ala 265 270 275 aag gct ctt gta tat tataca ttt gga gct ctt ggg ggc aat cta ata 921 Lys Ala Leu Val Tyr Tyr ThrPhe Gly Ala Leu Gly Gly Asn Leu Ile 280 285 290 gcc cac atg gtt ttg ggttac aga tac tgg gct ggc atc ggc gtc ctc 969 Ala His Met Val Leu Gly TyrArg Tyr Trp Ala Gly Ile Gly Val Leu 295 300 305 cag agt tgt gaa tct gccctg act cac tat cgt ctt gtt gcc aat cat 1017 Gln Ser Cys Glu Ser Ala LeuThr His Tyr Arg Leu Val Ala Asn His 310 315 320 gtt gct agt gat atc tcgcta aca gga ggc tca gta gta cag aga ata 1065 Val Ala Ser Asp Ile Ser LeuThr Gly Gly Ser Val Val Gln Arg Ile 325 330 335 340 cgg ctg cct gat gaagtg gaa aat cca gga atg aac agt gga atg cta 1113 Arg Leu Pro Asp Glu ValGlu Asn Pro Gly Met Asn Ser Gly Met Leu 345 350 355 gaa gaa gat ttg attcaa tat tac cag ttc cta gct gaa aaa ggt gat 1161 Glu Glu Asp Leu Ile GlnTyr Tyr Gln Phe Leu Ala Glu Lys Gly Asp 360 365 370 gta caa gca cag gttggt ctt gga caa ctg cac ctg cac gga ggg cgt 1209 Val Gln Ala Gln Val GlyLeu Gly Gln Leu His Leu His Gly Gly Arg 375 380 385 gga gta gaa cag aatcat cag aga gca ttt gac tac ttc aat tta gca 1257 Gly Val Glu Gln Asn HisGln Arg Ala Phe Asp Tyr Phe Asn Leu Ala 390 395 400 gca aat gct ggc aattca cat gcc atg gcc ttt ttg gga aag atg tat 1305 Ala Asn Ala Gly Asn SerHis Ala Met Ala Phe Leu Gly Lys Met Tyr 405 410 415 420 tcg gaa gga agtgac att gta cct cag agt aat gag aca gct ctc cac 1353 Ser Glu Gly Ser AspIle Val Pro Gln Ser Asn Glu Thr Ala Leu His 425 430 435 tac ttt aag aaagct gct gac atg ggc aac cca gtt gga cag agt ggg 1401 Tyr Phe Lys Lys AlaAla Asp Met Gly Asn Pro Val Gly Gln Ser Gly 440 445 450 ctt gga atg gcctac ctc tat ggg aga gga gtt caa gtt aat tat gat 1449 Leu Gly Met Ala TyrLeu Tyr Gly Arg Gly Val Gln Val Asn Tyr Asp 455 460 465 cta gcc ctt aagtat ttc cag aaa gct gct gaa caa ggc tgg gtg gat 1497 Leu Ala Leu Lys TyrPhe Gln Lys Ala Ala Glu Gln Gly Trp Val Asp 470 475 480 ggg cag cta cagctt ggt tcc atg tac tat aat ggc att gga gtc aag 1545 Gly Gln Leu Gln LeuGly Ser Met Tyr Tyr Asn Gly Ile Gly Val Lys 485 490 495 500 aga gat tataaa cag gcc ttg aag tat ttt aat tta gct tct cag gga 1593 Arg Asp Tyr LysGln Ala Leu Lys Tyr Phe Asn Leu Ala Ser Gln Gly 505 510 515 ggc cat atcttg gct ttc tat aac cta gct cag atg cat gcc agt ggc 1641 Gly His Ile LeuAla Phe Tyr Asn Leu Ala Gln Met His Ala Ser Gly 520 525 530 acc ggc gtgatg cga tca tgt cac act gca gtg gag ttg ttt aag aat 1689 Thr Gly Val MetArg Ser Cys His Thr Ala Val Glu Leu Phe Lys Asn 535 540 545 gta tgt gaacga ggc cgt tgg tct gaa agg ctt atg act gcc tat aac 1737 Val Cys Glu ArgGly Arg Trp Ser Glu Arg Leu Met Thr Ala Tyr Asn 550 555 560 agc tat aaagat ggc gat tac aat gct gca gtg atc cag tac ctc ctc 1785 Ser Tyr Lys AspGly Asp Tyr Asn Ala Ala Val Ile Gln Tyr Leu Leu 565 570 575 580 ctg gctgaa cag ggc tat gaa gtg gca caa agc aat gca gcc ttt att 1833 Leu Ala GluGln Gly Tyr Glu Val Ala Gln Ser Asn Ala Ala Phe Ile 585 590 595 ctt gatcag aga gaa gca agc att gta ggt gag aat gaa act tat ccc 1881 Leu Asp GlnArg Glu Ala Ser Ile Val Gly Glu Asn Glu Thr Tyr Pro 600 605 610 aga gctttg cta cat tgg aac agg gcc gcc tct caa ggc tat act gtg 1929 Arg Ala LeuLeu His Trp Asn Arg Ala Ala Ser Gln Gly Tyr Thr Val 615 620 625 gct agaatt aag ctc gga gac tac cat ttc tat ggg ttt ggc acc gat 1977 Ala Arg IleLys Leu Gly Asp Tyr His Phe Tyr Gly Phe Gly Thr Asp 630 635 640 gta gattat gaa act gca ttt att cat tac cgt ctg gct tct gag cag 2025 Val Asp TyrGlu Thr Ala Phe Ile His Tyr Arg Leu Ala Ser Glu Gln 645 650 655 660 caacac agt gca caa gct atg ttt aat ctg gga tat atg cat gag aaa 2073 Gln HisSer Ala Gln Ala Met Phe Asn Leu Gly Tyr Met His Glu Lys 665 670 675 ggactg ggc att aaa cag gat att cac ctt gcg aaa cgt ttt tat gac 2121 Gly LeuGly Ile Lys Gln Asp Ile His Leu Ala Lys Arg Phe Tyr Asp 680 685 690 atggca gct gaa gcc agc cca gat gca caa gtc cca gtc ttc tta gcc 2169 Met AlaAla Glu Ala Ser Pro Asp Ala Gln Val Pro Val Phe Leu Ala 695 700 705 ctctgc aaa ttg ggc gtc gtc tat ttc ttg cag tac ata cgg gaa aca 2217 Leu CysLys Leu Gly Val Val Tyr Phe Leu Gln Tyr Ile Arg Glu Thr 710 715 720 aacatt cga gat atg ttc acc caa ctt gat atg gac cag ctt ttg gga 2265 Asn IleArg Asp Met Phe Thr Gln Leu Asp Met Asp Gln Leu Leu Gly 725 730 735 740cct gag tgg gac ctt tac ctc atg acc atc att gcg ctg ctg ttg gga 2313 ProGlu Trp Asp Leu Tyr Leu Met Thr Ile Ile Ala Leu Leu Leu Gly 745 750 755aca gtc ata gct tac agg caa agg cag cac caa gac atg cct gca ccc 2361 ThrVal Ile Ala Tyr Arg Gln Arg Gln His Gln Asp Met Pro Ala Pro 760 765 770agg cct cca ggg cca cgg cca gct cca ccc cag cag gag ggg cca cca 2409 ArgPro Pro Gly Pro Arg Pro Ala Pro Pro Gln Gln Glu Gly Pro Pro 775 780 785gag cag cag cca cca cag taataggcac tgggtccagc cttgatcagt 2457 Glu GlnGln Pro Pro Gln 790 gacagcgaag gaagttatct gctgggaaca cttgcatttgatttaggacc ttggatcagt 2517 ggtcacctcc cagaagaggc acggcacaag gaagcattgaattcctaaag ctgcttagaa 2577 tctgatgcct ttattttcag ggataagtaa ctcttacctaaactgagctg aatgtttgtt 2637 tcagtgccat atggagtaac aactttcagt ggcttttttttttcttttct ggaaacatat 2697 gtgagacact cagagtaatg tctactgtat ccagctatctttctttggat ccttttggtc 2757 attatttcag tgtgcataag ttcttaatgt caaccatctttaaggtattg tgcatcgaca 2817 ctaaaaactg atcagtgtta aaaaggaaaa cccagttgcaagtttaaacg tgttcgaaag 2877 tctgaaaata gaacttgcct tttaagttaa aaaaaaaaaaaaagctatct tgaaaatgtt 2937 ttggaactgc gataactgag aaacttctta ccagtccacatgcaattaaa catattcagc 2997 atatttgtta ttttaaaagg gagggttggg aggtttcttattggtgattg tcacacggta 3057 taccatactc ctctccttca aagaatgaaa ggccttgttaaggagttttt tgtgagcttt 3117 acttctttgg aatggaatat acttatgcaa aaccttgtgaactgactcct tgcactaacg 3177 cgagtttgcc ccacctactc tgtaatttgc ttgtttgttttgaatataac agagccttga 3237 tccagaagcc agaggatgga ctaagtggga gaaattagaaaacaaaacga actctggttg 3297 gggtactacg atcacagaca cagacatact tttcctaaagttgaagcatt tgttcccagg 3357 atttatttta ctttgcattt ctttttgcac aaagaacacatcaccttcct gaattcttta 3417 aatatgaaat atcattgcca gggtatggct tacagtgactactattatca atactaaaac 3477 tcagagaatc aaagatggat taaactcagt ggttgatgaaagccaaaacc tgtttgtact 3537 gttctatact attcaggtat ctttttattt ctgatagttttatattataa tagaaagcca 3597 gccactgctt agctatcata gtcaccattt tctcactgttaacattagga aaatcaaggc 3657 tactatgctt caggattgtc tggttaaata gtatgggaaaaaaactgaag agtttcaaca 3717 taattacaca cgtgaaataa ttacagctta aactgaatttgtatttcatt ttattgtcag 3777 atggtggtgt tcaccagcct gtatcttgtc tgagactgcattcgtatctg agcaggtttt 3837 ctatgcctac tgatgtcagt atgtttatac taaccttcatgcttttttcc cagaatccct 3897 catctgccag aaaacttgaa aagtttattg cttgtagagttgtactgctt tgatttttga 3957 agttggggta gtagttagaa ctagatttaa ctagtctataatgaacatga aggcttttat 4017 atatgaagtt gtataccttt ttgtgtttag agaattatgggaaacctggt aagcaaaact 4077 ttcctcccag ataattgctt ccaaattcga agagttagtcaccaagagag ccatatgtat 4137 gaaagcgtat ctgtgaaagg taggaaactt accccccctaagtgtaatgt tgctttaggc 4197 aactcttgta aatagtgaga cttgtttggt ctcttacatgtagagatttg agtgcagttg 4257 gtacagtact ttggtgtctc caccactgtc ccttctccccgcttcaaaat aagtgtaatc 4317 cacggtagca gccacacttc cttcagaagg aactgttataatttatttaa aagttgaaaa 4377 accacccaag atgactacca actttcactt tttttcttctgccatccacc ctcattttcc 4437 ctttagcaag atttttatat ctaactttcc ttccctccattgagtacgtg ctttgagaaa 4497 acatttctta aaacagtgtg tgccacctaa ggctggatgggaaagtgcag tcttgttgtt 4557 catataaaaa acacacttct tattagttta cccacttgcctttttctatt gttaatgttc 4617 tgaatttcct tttcttggct tgtttctact tcattttaaccctgggtcac ttgctgccag 4677 cagtttgtga atggtgtctt tcaaataact tagttcttatggcttcactt aaagactgtc 4737 tcaaaaatac tttgctctct tcttcttttt tgttcatgggacatggtacc taagcaaata 4797 ggagttgggt ttggtttttc tcctaaaata atgctcaatacttacctaat caaatggcat 4857 ccatttgaat aaaatgacaa taactaaagc tagttaatgtcagtgacatt aaactaactc 4917 caggattcag gagttttaat gttagaattt agatttaacagatagagtgt ggcttcattt 4977 gtccatggta gcccatctct cctaagacct tttctagtctgtcttcctgc cttcgaactt 5037 gatgacagta aaaccctgtt tagtattctc ttgtgcatttggtttgttgg ttagccgact 5097 gtcttgaaac tattcatttt gcttctagtt ttattttacagaggtagcat tggtgggttt 5157 tttttttttt ttctgtctct gtgtttgaag tttcagtttctgttttctag gtaaggctta 5217 tttttgatta gcagtcaatg gcaaagaaaa agtaaatcaaagatgacttc ttttcaaaat 5277 gtatggccct tttattgcac ttttaactca gatgaatttataaattatta atcttgatac 5337 taaggatttg ttactttttt gcatattagg ttaatttttaccttacatgt gagagtctta 5397 ccactaagcc attctgtctc tgtactgttg ggaagttttggaaacccctg ccagtgatct 5457 ggtgatgatc tgatgattta tttaaagagc cgttgatgcctccaggaaac ttaagtattt 5517 tattaatata tatataggaa ttttttttta ttttgctttgtctttctctc ccttctttta 5577 tcctcatgtt cattcttcaa accagtgttt tggaagtatgcatgcaggcc tataaatgaa 5637 aaacacaatt ctttatgtgt atagcatgtg tattaatgtctaactacata cgcaaaaact 5697 tcctttacag aggttcggac taacatttca catgcacatttcaaaacaag atgtgtcatg 5757 aaaacagccc ctttacctgc caagacaagc agggctatatttcagtgaca gctggatatt 5817 ttgtttctga aagtgaatct cataatatat atatgtattacacattatta tgactagaag 5877 tatgtaagaa atgatcagaa caaaagaaaa tttctattttcatgcaaata tttttcatca 5937 gtcatcactc tcaaatataa attaaaatat aacactcctgaatgcctgag gcacgatctg 5997 gattttaaat gtgtggtatt cattgaaaag aagctctccacccacttggt atttcaagaa 6057 aatttaaaac gatcccaagg aaagatgatt tgtatgttaaagtgactgca caagtaaaag 6117 tccaatgttg tgtgcatgaa aaggattcct tggttatgtgcagggaatca tctcacatgc 6177 tgtttttcct atttggtttg agaaacaggc tgacactattctctttgatt agaaaataaa 6237 ctcataaaac tcataatgtt gatataatca agatgttaaccactataaat atgtagaaga 6297 ggaagtttta aatagacctt aagctggcat tgtgaaggaacaccatggta gactcttttt 6357 ggtaatggta ttttgtattt aatgaaatgc agtataaaggttggtgaagt gtaataataa 6417 ttgtgtaaac aaatcctgtt taatagaaga gatgtacagaatcgttttgg tactgtatct 6477 tgaaacttgt gaaataaaga ttccactttt ggttatcctgtatgctgtaa tataccacaa 6537 ccaagcaccc tttccagaca gacttttttt aagctgaatgaatccaattt tttaatgttt 6597 tttggaaatt cagaagcttc tgaaaacatt cacttgtggcaatttgaatt tatctttcat 6657 tttaaactcc tgaaattcag atttttacaa gtccaatattgccctaggga gaacatgaat 6717 ttgctaagaa atgttatctt ttaaatctct gatatctttgtcttgaagca gccttgatat 6777 gtagtaagcg tgattcactt tagcctgatt ataatattatttatctaaag tttgtttatg 6837 cattgccttg tcccaggaat tttttaagag gacttgcagagacacgtacc acacagtaac 6897 atttagacta aatatgctct gagtaaagga gaaatgaaaaaatattaaat caagagtgaa 6957 catgtacaca aagtgcaatt ggaagtgggc tacaaatttagcccccagct tcccagcagg 7017 caactcaaag aggtaactga ggtaaaatgt tccagctcagaagcattgga tcttggataa 7077 aaagcctaca tgatgcaaac tgtggcaact gagatgtcagatctcaagat ctcaaattgt 7137 acttgtggga gcacagtcag tgaccccaga tgaccttgactgacctaaaa gttgtggggg 7197 aagtcggatg tcagagcctt aacaccagca ggtgaccatccaacctgggg caatgcctgc 7257 ctgttcacca cttagcctct ttctggcaag tcattagaatgtcctccatc ttcattggct 7317 gcaacttgat gagctacagc ctctttccta acttcctttatgatgctagt ttaggttggt 7377 tataccagct tggaagtatg cttagattaa gttacagcagatacacaaat tagatgcaag 7437 taaaaaaaat cagaatttct gtagtagaaa ctacgaaaaataaaaaggaa agtttttact 7497 ttttgggtat ttttttacga ataagaaaaa gtgagcgttaatcagttcaa aaggaggtac 7557 tgctgtgtaa tgggctttgt acgttccttc tcatgtcacttacgtcacta cttcgccatc 7617 aaattgaaca agcttttaat tagatcctga aaattcactatgctagtagt ttattggtag 7677 tattatattt tgagtagaac tctgattttc cctagaggccaaattctttt tatctgggtt 7737 aatttctttt aaacataaca atgttaatgc tgaattgtatattaaatccc atttctaaaa 7797 accacacaat tttttctcat gtaagttgag tggaatgtggttagttaact gaatttggaa 7857 tgttcatata aataatttgt tgctgctc 7885 <210> SEQID NO 4 <211> LENGTH: 10 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <221> NAME/KEY: misc_feature <223> OTHERINFORMATION: Primer Sequence for PCR of TSA305 <400> SEQUENCE: 4gatctgacac 10 <210> SEQ ID NO 5 <211> LENGTH: 28 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY:misc_feature <223> OTHER INFORMATION: Primer Sequence for PCR of TSA305<400> SEQUENCE: 5 gatcggatcc aggaggatgc gggtccgg 28 <210> SEQ ID NO 6<211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <221> NAME/KEY: misc_feature <223> OTHER INFORMATION:Primer Sequence for PCR of TSA305 <400> SEQUENCE: 6 gatcctcgagttactgtggt ggctgctgct 30

What is claimed is:
 1. An isolated polynucleotide which consists of anucleotide sequence coding for a protein having the amino-acid sequenceof SEQ ID NO:1.
 2. The polynucleotide as claimed in claim 1, whereinsaid polynucleotide consists of the nucleotide sequence of SEQ ID NO:2.3. An isolated polynucleotide selected from he group consisting of: (a)a polynucleotide consisting of SEQ ID NO:5; and (b) a polynucleotideconsisting of SEQ ID NO:6.